Enzymatic generation of lactulose in sweet and acid whey: Optimization of feed composition and structural elucidation of 1-lactulose.

Prebiotics are rising in interest in commercial scale productions due to increasing health awareness of consumers. Under bio-economic aspects, sweet and acid whey provide a suitable feed medium for the enzymatic generation of prebiotic lactulose. Since whey has a broad variation in composition, the influence of the feed composition on the concentration of generated lactulose was investigated. The influence of lactose and fructose concentration as well as enzymatic activity of two commercially available ß-galactosidases were investigated. The results were evaluated via response surface analysis with a quadratic model containing pairwise interaction terms. The optimal feed composition yielding a theoretical maximal amount of lactulose was determined as 1.28 or 0.74 mol/kg fructose and 0.17 or 0.19 mol/kg lactose with an enzymatic activity of 2.0 or 2.8 µkat/kg for acid (pH 4.4) or sweet (pH 6.6) whey. Furthermore, the major reaction product was isolated and subsequently, the structural identity was elucidated and verified via extensive NMR analysis.

Validation of UPLC-MS/MS Method for Determination of Urinary Lactulose/Mannitol.

Determination of urinary lactulose/mannitol is one of the most used tests to evaluate intestinal barrier function. High-performance liquid chromatography (HPLC) separation with electrospray ionization tandem mass spectrometry guarantees high levels of selectivity and reproducibility. In this paper we report an upgrade of the previous published liquid chromatography tandem mass spectrometry method, introducing more reliable internal standards and ultra-performance liquid chromatography with ethylene bridged hybrid amide columns. The ultra-performance liquid chromatography provided an efficient chromatographic separation of the two sugars in 5 min, compared to 15 min using the previous method. The limit of quantification was 10 µg/mL for mannitol and 2.5 µg/mL for lactulose, and the assay was linear up to 1000 µg/mL for mannitol and 1000 µg/mL for lactulose. The within-run precision and accuracy ranged from 0.7 to 2.9% and 97.2 to 101.2%, respectively. The between-run precision and accuracy ranged from 1.9 to 4.7% and 94.8 to 97.5%, respectively. Recovery was higher than 90.2% for both lactulose and mannitol, and the matrix effect for both lactulose and mannitol was lower than 15%. With this new method we have a real improvement in terms of accuracy and reproducibility, ensuring results in shorter time. The changes to the previous protocol make this method excellent for routine purposes.

Preparation of high-purity lactulose through efficient recycling of catalyst sodium aluminate and nanofiltration: a pilot-scale purification.

BACKGROUND: Lactulose, a valuable lactose-originated 'bifidus factor' product, is exclusively produced by chemical-based isomerization commercially. A complexing agent of sodium aluminate exhibiting high conversion efficiency and strong recyclable stability is more practical for industrial applications. In this study, efficient purification of high-purity lactulose through recycling of sodium aluminate and further desalination by nanofiltration (NF) was implemented on a pilot scale. RESULTS: Over 99.5% of the catalyst was prior recycled in the form of Al(OH)3 precipitate by pH-induced precipitation and centrifugation; residual aluminum was further absorbed by ion exchange resin to an acceptable level (≤10 mg kg-1 ). Subsequently, impurities (monosaccharides and NaCl) were ideally separated from lactulose syrup by NF based on their significant retention differences (lactulose 94.8-97.2% > lactose 86.2-93.5% > monosaccharides 36.3-48.7% > NaCl 9.5-31.1%). High-purity (>95%) lactulose was obtained with >90% yield in both constant and variable volume diafiltration (CVD and VVD) modes when the volume dilution ratio (Vc /Vf ) was 4.0 and 2.5 respectively. Both experimental and predicted results showed that the VVD mode was more water-saving in practice. CONCLUSION: This is the first trial purification of lactulose syrup from chemical isomerization of lactose catalyzed by sodium aluminate, and the applied methodology is a promising industrial-scale purification strategy. © 2018 Society of Chemical Industry.

Lactulose: production, purification and potential applications.

Lactulose a "bifidus factor" is composed of galactose and fructose, which can be produced by the isomerization of lactose. It is a prebiotic carbohydrate which stimulates the growth of health-promoting bacteria in the gastrointestinal tract, such as bifidobacteria and lactobacilli and at the same time inhibits growth of pathogenic bacteria such as Salmonella. It can also be used for the treatment of constipation, hepatic encephalopathy, tumour prevention, and to maintain blood glucose and insulin level. This review provides comprehensive information on the different techniques used for the production of lactulose, purification and analysis. Besides this mechanism of action and its potential applications in food and pharmaceutical industries have also been discussed.

Purification of lactulose from mixtures with lactose using pressurized liquid extraction with ethanol-water at different temperatures.

The viability of the purification of lactulose from a mixture with lactose [70:30 (w/w)] using pressurized liquid extraction (PLE) at 1500 psi for 30 min was studied. Different temperatures (from 40 to 130 degrees C) and proportions of ethanol:water (70:30, 80:20, 90:10, 95:5, and 100:0) as the extraction solvent were assayed. Lactose and lactulose were measured by gas chromatographic analysis as their trimethylsilyl derivatives. Data were fitted through multiple linear regressions to different quadratic models to describe both the extraction yield (in terms of mg of lactulose) and the purity of the lactulose extracted. The optimum extraction conditions provided by the model were as follows: extraction temperature, 40 degrees C; and solvent composition, 70:30 ethanol:water. PLE extraction under the optimized conditions was also applied to purify lactulose from lactose in a synthesis mixture. To our knowledge, this is the first time that PLE has been tested for extraction and purification of lactulose from its mixture with lactose; this technique showed several advantages over classical methods such as the short extraction time and the low solvent consumption.

A blood test for intestinal permeability and function: a new tool for the diagnosis of chronic intestinal disease in dogs.

We demonstrate that rhamnose, 3-O-methyl-D-glucose, D-xylose and lactulose may be quantified accurately in blood by HPLC and pulsed amperometric detection, thus enabling studies of intestinal permeability and function to be carried out using plasma samples. Prior to HPLC, the endogenous glucose was enzymatically modified to gluconic acid and the protein precipitated. The precision of the quantification of the sugars in plasma (CV: 2.2-5.7%; 8.7-10.6% at very low concentrations) compared well with the quantification in urine. The results for groups of 8 dogs with small intestinal bacterial overgrowth and 12 dogs with inflammatory bowel disease were shown to be significantly different from a group of 20 normal control dogs (P < 0.001), demonstrating the test's value as a diagnostic tool. The normal ranges in blood 2 h post oral administration were determined to be 0.05-0.17 for the lactulose/rhamnose ratio and 0.45-0.65 for the xylose/3-O-methylglucose ratio. This method may be employed advantageously when the collection of urine in intestinal permeability and function tests is difficult.

A novel HPLC method for the simultaneous quantification of monosaccharides and disaccharides used in tests of intestinal function and permeability.

The quantification of monosaccharides and disaccharides used as probes in intestinal function and permeability tests can be technically demanding, detracting from the value of this approach to the indirect assessment of intestinal damage. In this study, a procedure is described for the simultaneous quantification of rhamnose, lactulose, 3-O-methyl-D-glucose and xylose in urine by HPLC using an anion exchange column with pulsed amperometric detection. This method is relatively fast and simple to perform, requiring no pre-treatment of urine samples or post-column derivatization. Accuracy and precision of determinations are illustrated by analytical recoveries (mean percentage +/- S.D., CV., n = 30) for multiple batch analyses of a diluted urine sample containing 20 mg/l of rhamnose (100 +/- 6.8, 6.2%), lactulose (100 +/- 6.1, 5.5%), 3-O-methyl-D-glucose (98 +/- 5.9, 5.5%) and xylose (104 +/- 7.1, 6.5%). Linearity of standard curves indicated that the lower limit for accurate quantification was 0.1 mg/l for all four sugars. Urinary recoveries following oral administration of these sugars to control dogs were determined as a baseline for the investigation of intestinal damage in this species and comparison of chromatograms illustrated enhanced permeability in dogs with gluten-sensitive enteropathy.